Peptidoglycan from Akkermansia muciniphila MucT: chemical structure and immunostimulatory properties of muropeptides.

Akkermansia muciniphila is an intestinal symbiont known to improve the gut barrier function in mice and humans. Various cell envelope components have been identified to play a critical role in the immune signaling of A. muciniphila, but the chemical composition and role of peptidoglycan (PG) remained elusive. Here, we isolated PG fragments from A. muciniphila MucT (ATCC BAA-835), analyzed their composition and evaluated their immune signaling capacity. Structurally, the PG of A. muciniphila was found to be noteworthy due of the presence of some nonacetylated glucosamine residues, which presumably stems from deacetylation of N-acetylglucosamine. Some of the N-acetylmuramic acid (MurNAc) subunits were O-acetylated. The immunological assays revealed that muropeptides released from the A. muciniphila PG could both activate the intracellular NOD1 and NOD2 receptors to a comparable extent as muropeptides from Escherichia coli BW25113. These data challenge the hypothesis that non-N-acetylattion of PG can be used as a NOD-1 evasion mechanism. Our results provide new insights into the diversity of cell envelope structures of key gut microbiota members and their role in steering host-microbiome interactions.

Structure of the O-Antigen and the Lipid†A from the Lipopolysaccharide of Fusobacterium nucleatum ATCC 51191.

Fusobacterium nucleatum is a common member of the oral microbiota. However, this symbiont has been found to play an active role in disease development. As a Gram-negative bacterium, F. nucleatum has a protective outer membrane layer whose external leaflet is mainly composed of lipopolysaccharides (LPSs). LPSs play a crucial role in the interaction between bacteria and the host immune system. Here, we characterised the structure of the O-antigen and lipid A from F. nucleatum ssp. animalis ATCC 51191 by using a combination of GC-MS, MALDI and NMR techniques. The results revealed a novel repeat of the O-antigen structure of the LPS, [→4)-ß-d-GlcpNAcA-(1→4)-ß-d-GlcpNAc3NAlaA-(1→3)-α-d-FucpNAc4NR-(1→], (R=acetylated 60 %), and a bis-phosphorylated hexa-acylated lipid A moiety. Taken together these data showed that F. nucleatum ATCC 51191 has a distinct LPS which might differentially influence recognition by immune cells.

Carbohydrate-based adjuvants.

Carbohydrate adjuvants are safe and biocompatible compounds usable as sustained delivery systems and stimulants of ongoing humoral and cellular immune responses, being especially suitable for the development of vaccines against intracellular pathogens where alum is useless. The development of new adjuvants is difficult and expensive, however, in the last two years, seven new carbohydrate-based adjuvants have been patented, also there are twelve ongoing clinical trials of vaccines that contain carbohydrate-based adjuvants, as well as numerous publications on their mechanism of action and safety. More research is necessary to improve the existent adjuvants and develop innovative ones.

Adherence to clinical trial registration in countries of Latin America and the Caribbean, 2015.

OBJECTIVE: To determine the prevalence of clinical trial registration in the International Clinical Trial Registry Platform (ICTRP) for studies from Latin America and the Caribbean (LAC) and to identify the key characteristics that lead to prospective and retrospective registration. METHODS: A cross-sectional study identified published, clinical trial studies through a search of PubMed, LILACS (Latin American and Caribbean Center on Health Sciences Information), and the Cochrane Central Register of Controlled Trials. Studies were included if published on 1 January - 31 December 2015, at least one author was affiliated with at least one LAC country, the clinical trial was conducted in at least one LAC site, and the full text of the article was available. A manual search of reference lists was also conducted. ICTRP registration information and key trial characteristics were compared. RESULTS: Of 1 502 CT references that met inclusion criteria, 297 were randomly-selected, 90.9% of which were published in English, 65% from Brazil, and 76.8% had a LAC author as the first author. The proportion of CT registered in the ICTRP was 59.9 %, of which 51.7% were registered prospectively. Clinicaltrials.gov was most frequently used registry (84.8%), followed by the Registro Brasileiro de Ensaios Clínicos and the Registro Público Cubano de Ensayos Clínicos. Key characteristics that favored registration were being in study phase 3 or 4 or being a multi-center study. Data was compared to a similar study from 2013 that reported a registration rate of only 19.8%. CONCLUSIONS: Registration adherence and prospective registration have increased in LAC in recent years, but the proportion of unregistered CT remains high. While there are still many challenges to overcome, the adherence strategies implemented in recent years have proven effective.

Extensively Antibiotic-Resistant Bacterial Infections in Trauma Cases Managed at the Médecins Sans Frontières Tertiary Orthopaedic Center in Mosul, Iraq: A Case Series.

The Médecins Sans Frontières Tertiary Orthopaedic Care center in Mosul, Iraq, provides reconstructive surgery, microbiological analysis, integrated infection prevention and control, and antibiotic stewardship services. Between May 2018 and February 2020, we recorded soft tissue and/or bone infections caused by gram-negative extensively drug-resistant (XDR) bacteria in 4.9% (13/266) of the admitted patients. The XDR bacteria identified among 12 patients in this case series were extended-spectrum ß-lactamase-producing Klebsiella pneumoniae (n = 5, 41.7%) with intermediate sensitivity or resistance to imipenem and/or meropenem, Acinetobacter spp (n = 3, 25.0%; 2 Acinetobacter baumannii strains) resistant to imipenem and/or meropenem, Pseudomonas aeruginosa (n = 2, 16.7%) resistant to imipenem and meropenem, and extended-spectrum ß-lactamase-producing Proteus mirabilis (n = 2, 16.7%) resistant to meropenem. Most XDR isolates were sensitive only to colistin or polymyxin B, neither of which is available in Iraq. Therefore, the only treatment option was multiple rounds of surgical debridement and wound care. The infection was deemed cured before discharge in 7 patients (58.3%). Meanwhile, 4 patients (33.3%) were discharged with unhealed wounds, and outpatient follow-up was planned. One patient died in the intensive care unit of a referral hospital after developing septicemia postsurgery. XDR bacteria pose substantial health risks in Iraq. Thus, improving antimicrobial stewardship and accessibility to essential antibiotics is critical to address this issue.

The role of bacterial vaccines in the fight against antimicrobial resistance: an analysis of the preclinical and clinical development pipeline.

Vaccines can be highly effective tools in combating antimicrobial resistance as they reduce infections caused by antibiotic-resistant bacteria and antibiotic consumption associated with disease. This Review looks at vaccine candidates that are in development against pathogens on the 2017 WHO bacterial priority pathogen list, in addition to Clostridioides difficile and Mycobacterium tuberculosis. There were 94 active preclinical vaccine candidates and 61 active development vaccine candidates. We classified the included pathogens into the following four groups: Group A consists of pathogens for which vaccines already exist-ie, Salmonella enterica serotype Typhi, Streptococcus pneumoniae, Haemophilus influenzae type b, and M tuberculosis. Group B consists of pathogens with vaccines in advanced clinical development-ie, extra-intestinal pathogenic Escherichia coli, Salmonella enterica serotype Paratyphi A, Neisseria gonorrhoeae, and C difficile. Group C consists of pathogens with vaccines in early phases of clinical development-ie, enterotoxigenic E coli, Klebsiella pneumoniae, non-typhoidal Salmonella, Shigella spp, and Campylobacter spp. Finally, group D includes pathogens with either no candidates in clinical development or low development feasibility-ie, Pseudomonas aeruginosa, Acinetobacter baumannii, Staphylococcus aureus, Helicobacter pylori, Enterococcus faecium, and Enterobacter spp. Vaccines are already important tools in reducing antimicrobial resistance and future development will provide further opportunities to optimise the use of vaccines against resistance.

Dissecting Lipopolysaccharide Composition and Structure by GC-MS and MALDI Spectrometry.

Lipopolysaccharides (LPSs) are the main components of the external leaflet of the outer membrane of Gram-negative bacteria. They exert multiple functions, starting from conferring stability to the bacterial membrane to mediating the interaction of the microbe with the external environment. The composition and the structure of LPSs present tremendous diversity even within bacteria of the same species, and for this reason, the determination of the structure of these molecules is crucial because it can provide information on the motifs key for the virulence of a pathogen or that are associated to a bacterium of the commensal or beneficial microbiota. In addition, structural data disclose the effects triggered from a mutation or from the use of an antibiotic, or they can be used as tools to check the quality of adjuvants and/or medications, as vaccines, that make use of LPS.The structural study of LPSs is complex, and it can be achieved with the right combination of different techniques. In this frame, this chapter focuses on the two MS-based approaches, the gas chromatography-mass spectrometry (GC-MS) and the matrix-assisted laser desorption/ionization (MALDI).

Preparing for future challenges in risk assessment in the European Union.

In light of the new EU policy targets (e.g., Farm to Fork strategy) and the revised legal framework (Transparency Regulation), the European Food Safety Authority (EFSA) needs to invest further in preparedness in regulatory and communication science for food safety. To achieve this, EFSA has established a process of advancing selected scientific themes to anticipate future challenges.

Lipopolysaccharide lipid A: A promising molecule for new immunity-based therapies and antibiotics.

Lipopolysaccharides (LPS) are the main components of the external leaflet of the Gram-negative outer membrane and consist of three different moieties: lipid A, core oligosaccharide, and O-polysaccharide. The lipid A is a glucosamine disaccharide with different levels of acylation and phosphorylation, beside carrying, in certain cases, additional substituents on the sugar backbone. It is also the main immunostimulatory part of the LPS, as its recognition by the host immune system represents a fundamental event for detection of perilous microorganisms. Moreover, an uncontrolled immune response caused by a large amount of circulating LPS can lead to dramatic outcomes for human health, such as septic shock. The immunostimulant properties of an LPS incredibly vary depending on lipid A chemical structure, and for this reason, natural and synthetic variants of the lipid A are under study to develop new drugs that mimic or antagonise its natural effects. Here, we review past and recent findings on the lipid A as an antibiotic target and immune-therapeutic molecule, with a special attention on the crucial role of the chemical structure and its exploitation for conceiving novel strategies for treatment of several immune-related pathologies.

Liquid-state NMR spectroscopy for complex carbohydrate structural analysis: A hitchhiker's guide.

Structural determination of carbohydrates is mostly performed by liquid-state NMR, and it is a demanding task because the NMR signals of these biomolecules explore a rather narrow range of chemical shifts, with the result that the resonances of each monosaccharide unit heavily overlap with those of others, thus muddling their punctual identification. However, the full attribution of the NMR chemical shifts brings great advantages: it discloses the nature of the constituents, the way they are interconnected, in some cases their absolute configuration, and it paves the way to other and more sophisticated analyses. The purpose of this review is to provide a practical guide into this challenging subject. It will drive through the strategy used to assign the NMR data, pinpointing the core information disclosed from each NMR experiment, and suggesting useful tricks for their interpretation, along with other resources pivotal during the study of these biomolecules.

Human Cryptic Host Defence Peptide GVF27 Exhibits Anti-Infective Properties against Biofilm Forming Members of the Burkholderia cepacia Complex.

Therapeutic solutions to counter Burkholderia cepacia complex (Bcc) bacteria are challenging due to their intrinsically high level of antibiotic resistance. Bcc organisms display a variety of potential virulence factors, have a distinct lipopolysaccharide naturally implicated in antimicrobial resistance. and are able to form biofilms, which may further protect them from both host defence peptides (HDPs) and antibiotics. Here, we report the promising anti-biofilm and immunomodulatory activities of human HDP GVF27 on two of the most clinically relevant Bcc members, Burkholderia multivorans and Burkholderia cenocepacia. The effects of synthetic and labelled GVF27 were tested on B. cenocepacia and B. multivorans biofilms, at three different stages of formation, by confocal laser scanning microscopy (CLSM). Assays on bacterial cultures and on human monocytes challenged with B. cenocepacia LPS were also performed. GVF27 exerts, at different stages of formation, anti-biofilm effects towards both Bcc strains, a significant propensity to function in combination with ciprofloxacin, a relevant affinity for LPSs isolated from B. cenocepacia as well as a good propensity to mitigate the release of pro-inflammatory cytokines in human cells pre-treated with the same endotoxin. Overall, all these findings contribute to the elucidation of the main features that a good therapeutic agent directed against these extremely leathery biofilm-forming bacteria should possess.

Siglec-7 Mediates Immunomodulation by Colorectal Cancer-Associated Fusobacterium nucleatum ssp. animalis.

Fusobacterium nucleatum is involved in the development of colorectal cancer (CRC) through innate immune cell modulation. However, the receptors of the interaction between F. nucleatum ssp. and immune cells remain largely undetermined. Here, we showed that F. nucleatum ssp. animalis interacts with Siglecs (sialic acid-binding immunoglobulin-like lectins) expressed on innate immune cells with highest binding to Siglec-7. Binding to Siglec-7 was also observed using F. nucleatum-derived outer membrane vesicles (OMVs) and lipopolysaccharide (LPS). F. nucleatum and its derived OMVs or LPS induced a pro-inflammatory profile in human monocyte-derived dendritic cells (moDCs) and a tumour associated profile in human monocyte-derived macrophages (moMϕs). Siglec-7 silencing in moDCs or CRISPR-cas9 Siglec-7-depletion of U-937 macrophage cells altered F. nucleatum induced cytokine but not marker expression. The molecular interaction between Siglec-7 and the LPS O-antigen purified from F. nucleatum ssp. animalis was further characterised by saturation transfer difference (STD) NMR spectroscopy, revealing novel ligands for Siglec-7. Together, these data support a new role for Siglec-7 in mediating immune modulation by F. nucleatum strains and their OMVs through recognition of LPS on the bacterial cell surface. This opens a new dimension in our understanding of how F. nucleatum promotes CRC progression through the generation of a pro-inflammatory environment and provides a molecular lead for the development of novel cancer therapeutic approaches targeting F. nucleatum-Siglec-7 interaction.

Antibiotic resistance patterns in human, animal, food and environmental isolates in Ghana: a review.

Many articles have been published on resistant microorganisms isolated from humans, animals, foods and the environment in Ghana. However, there are no reviews that summarize the information on the isolates and antibiotics tested so far in the country. This literature review was completed through "PubMed" and "Google Scholar" searches. We included publications from the period 1975-2015 with a laboratory-based methodology to determine antibiotic resistance of strains isolated in Ghana. In total, 60 articles were included in the analysis with 10% of the articles carrying out nationwide research on antibiotic resistance. The regions of Ghana with the highest published articles were Greater Accra (40%), Ashanti (21.7%) and Northern Region (10%). Most of the studies (86.7%) were related to isolates collected from human samples followed by environmental (5%), animal (3%) and food samples (2%). Ten different bacteria genera were observed in the studies. The most common was Escherichia coli, followed by Staphylococcus spp., Mycobacterium spp. and Streptococcus spp. The highest mean resistance rate was encountered in Escherichia coli (62.2%) followed by Klebsiella spp. (60.4%) and Pseudomonas spp. (52.1%). High resistance rates have been found in Ghana, however, the data are skewed and some regions of the country have been neglected. There is a need for higher quality research to establish and monitor resistance patterns in Upper West, Brong-Ahafo, Volta and Eastern Regions of Ghana.

A cross-sectional study on antibiotic prescription in a teaching hospital in Ghana.

INTRODUCTION: Antibiotic misuse is the paramount factor for antibiotic resistance. Tamale Teaching Hospital (TTH), located in Ghana's Northern Region, is the biggest tertiary hospital in the Northern half of the country and consequently one of the biggest prescribers of antibiotics. Understanding the use of antibiotics in the TTH and providing information that could be inferred to develop strategies for antibiotic prescription is of extreme importance in this era of multiple and pan-resistant strains of pathogenic microorganisms. METHODS: A cross-sectional study on the use of antibiotics at TTH in the Northern region of Ghana was performed. Data were collected by reviewing 10% of patients' files from January to June 2015 and then assessed for its appropriateness against the criteria based on the British National Formulary (BNF) 2015 and BNF children 2013-2014. Results were expressed in frequencies and percentages. RESULTS: A total of 617 patients' records were included in this study. Up to 385 cases of different antibiotic misuse were found, comprising of 335 errors in prescriptions and 50 non-completed treatments. The most common prescription error was made on treatment duration (29.6%). The potential interactions were 16.7%. CONCLUSION: The study revealed a high burden of antibiotics misuse in TTH. This suggests a need for the development of an antibiotic stewardship programme for the hospital.

Structural features and immunological perception of the cell surface glycans of Lactobacillus plantarum: a novel rhamnose-rich polysaccharide and teichoic acids.

The capsular material from Lactobacillus plantarum IMB19, an isolate from fermented vegetables, has been analyzed and our results demonstrate that most of the coat of this bacterium consists of glycerol- and ribitol-type teichoic acids, further decorated with other substituents (α-glucose and alanine), and of a capsular polysaccharide (CPS) with a linear nonasaccharide repeating unit, rich in rhamnose, interconnected to the next via a phosphodiester bridge. Stimulation of immune cells with the total capsular material resulted in the enhancement of immunostimulatory (IFNγ, TNF-α, IL-6 and IL-12) or immuno-regulatory (IL-10) cytokines in an in vitro splenocyte culture system. The capsular polysaccharide, and not the teichoic acids mixture, was responsible for the IFNγ production. Indeed, a significant increase of IFNγ along with other inflammatory cytokines, and a dose response in IFNγ expression with an EC50 of 3.16 µM was found for CPS, disclosing that this polysaccharide is a potent immunostimulatory molecule.

Adherence to clinical trial registration in countries of Latin America and the Caribbean, 2015 / Cumplimiento del registro de ensayos clínicos en los países de América Latina y el Caribe, 2015 / Adesão ao registro de ensaios clínicos em países da América Latina e Caribe, 2015

ABSTRACT Objective To determine the prevalence of clinical trial registration in the International Clinical Trial Registry Platform (ICTRP) for studies from Latin America and the Caribbean (LAC) and to identify the key characteristics that lead to prospective and retrospective registration. Methods A cross-sectional study identified published, clinical trial studies through a search of PubMed, LILACS (Latin American and Caribbean Center on Health Sciences Information), and the Cochrane Central Register of Controlled Trials. Studies were included if published on 1 January - 31 December 2015, at least one author was affiliated with at least one LAC country, the clinical trial was conducted in at least one LAC site, and the full text of the article was available. A manual search of reference lists was also conducted. ICTRP registration information and key trial characteristics were compared. Results Of 1 502 CT references that met inclusion criteria, 297 were randomly-selected, 90.9% of which were published in English, 65% from Brazil, and 76.8% had a LAC author as the first author. The proportion of CT registered in the ICTRP was 59.9 %, of which 51.7% were registered prospectively. Clinicaltrials.gov was most frequently used registry (84.8%), followed by the Registro Brasileiro de Ensaios Clínicos and the Registro Público Cubano de Ensayos Clínicos. Key characteristics that favored registration were being in study phase 3 or 4 or being a multi-center study. Data was compared to a similar study from 2013 that reported a registration rate of only 19.8%. Conclusions Registration adherence and prospective registration have increased in LAC in recent years, but the proportion of unregistered CT remains high. While there are still many challenges to overcome, the adherence strategies implemented in recent years have proven effective.

RESUMEN Objetivo Determinar la prevalencia del registro de ensayos clínicos de América Latina y el Caribe en la Plataforma de Registros Internacionales de Ensayos Clínicos (ICTRP, por su sigla en inglés) y definir los elementos clave que fomentan el registro prospectivo y retrospectivo de estudios. Métodos Se realizó un estudio transversal para encontrar los ensayos clínicos publicados mediante una búsqueda en PubMed, LILACS (Centro Latinoamericano y del Caribe para Información en Ciencias de la Salud) y el Registro Central Cochrane de Ensayos Clínicos Controlados. Se incluyeron los estudios que habían sido publicados entre el 1 de enero y el 31 de diciembre del 2015, que tenían cuando menos un autor afiliado a uno o más países de América Latina y el Caribe, que se habían realizado al menos en un centro de América Latina y el Caribe, y que tenían el texto completo del artículo disponible. También se llevó a cabo una búsqueda manual en listas de referencia. Se comparó la información sobre registros de la ICTRP y las características clave de los ensayos clínicos. Resultados De las 1 502 referencias que cumplieron los criterios de inclusión, se seleccionaron 297 aleatoriamente. De estas, 90,9% se habían publicado en inglés, 65% eran de Brasil y 76,8% tenían como primer autor un investigador de América Latina y el Caribe. La proporción de ensayos clínicos registrados en la ICTRP fue de 59,9%, de los cuales 51,7% se habían registrado prospectivamente. Clinicaltrials.gov fue el registro usado con mayor frecuencia (84,8%), seguido por el Registro Brasileiro de Ensaios Clínicos y el Registro Público Cubano de Ensayos Clínicos. Se determinó que las características clave que favorecían el registro eran que fuese un estudio de fase 3 o 4 o un estudio multicéntrico. Se compararon los datos con un estudio similar del 2013 en el que se había informado que la tasa de registro era de apenas 19,8%. Conclusiones En América Latina y el Caribe se ha observado en los últimos años un aumento en el cumplimiento del registro y del registro prospectivo de ensayos clínicos, pero la proporción de estudios sin registrar sigue siendo alta. Sin embargo, aunque persisten muchos retos que se deben superar, las estrategias adoptadas en los últimos años para que se cumpla este requisito han sido eficaces.

RESUMO Objetivo Determinar a prevalência do registro de estudos clínicos na Plataforma Internacional de Registro de Ensaios Clínicos (ICTRP) para estudos realizados na América Latina e Caribe (ALC) e identificar as principais características que conduzem ao registro prospectivo e retrospectivo. Métodos Em um estudo transversal, foram identificados os estudos clínicos publicados através de uma busca nas bases de dados PubMed, LILACS (Centro Latino-Americano e do Caribe de Informação em Ciências da Saúde) e Cochrane Central Register of Controlled Trials (CENTRAL). Foram incluídos estudos publicados de 1o. de janeiro a 31 de dezembro de 2015, em que pelo menos um dos autores provinha de um país da ALC, realizados em um ou mais centros na ALC e que apresentavam o texto completo disponível. Foi também feita uma busca manual das listas de referências. Foram comparados os dados sobre o registro na ICTRP e as principais características dos estudos. Resultados Das 1.502 referências de estudos clínicos que atenderam os critérios de inclusão, 297 foram selecionadas aleatoriamente. Verificou-se que 90,9% dos estudos foram publicados em inglês, 65% eram provenientes do Brasil e 76,8% tinham como primeiro autor um pesquisador da ALC. O percentual de registro dos estudos clínicos na ICTRP foi de 59,9%, sendo 51,7% registrado de forma prospectiva. Clinicaltrials.gov foi o registro mais usado (84,8%), seguido do Registro Brasileiro de Ensaios Clínicos e do Registro Público Cubano de Ensayos Clínicos. As principais características que contribuíram para o registro foram ser estudo de fase 3 ou 4 ou multicêntrico. Os dados foram comparados com um estudo semelhante realizado em 2013 que verificou uma taxa de registro de apenas 19,8%. Conclusões Houve um aumento na adesão ao registro e no registro prospectivo na ALC nos últimos anos, porém o percentual de estudos clínicos não registrados continua alto. Embora ainda existam muitos desafios a serem vencidos, as estratégias de adesão implementadas nos últimos anos têm sido eficazes.